DE1197423B - Process for the biochemical production of orotic acid - Google Patents

Description

FEDERAL REPUBLIC OF GERMANY GERMAN / fflt ^ S PATENT OFFICE

EDITORIAL

Inf. Α .:

Number:
File number:
Registration date:
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C12d

German class: 6b-16/02

1197423
K 43988IV a / 6 b
June 13, 1961
July 29, 1965

The invention relates to a process for the biochemical production of orotic acid by aerobic means Cultivation of a pyrimidine substance requiring mutant microorganisms in a conventional one Nutrient medium while maintaining a pH above 5.5.

Orotic acid is a precursor of pyrimidine compounds, which are constituents of nucleic acids, and is considered the same substance as vitamin B ₁₃ , which is important for the metabolism of nucleic acids, the multiplication of cells in the living body, heredity, protein synthesis and the metabolism of Saccharides and lipoids is. In recent studies on orotic acid, it has been found that it is remarkably effective in the treatment of liver diseases and has both hematopoietic and growth-promoting effects. Orotic acid is therefore becoming increasingly important.

It is known that mutants of Neurospora crassa requiring pyrimidines have a small amount Accumulate orotic acid in the culture medium (Journ. Of Biological Chemistry, 172, p. 525, 1948). It was however, no technically viable process for the production of orotic acid by fermentation has yet been proposed.

According to the invention a method for biochemical Production of orotic acid by aerobic cultivation of a pyrimidine substance requiring Microorganism mutants in a nutrient medium customary for this purpose while maintaining a pH value of over 5.5 proposed, which consists in the fact that the microorganism mutants Micrococcus glutamicus (534-615-305) ATCC 14275, micrococcus glutamicus (588-208) ATCC 14276 or Brevibacterium ammoniagenes ATCC 6871 used.

In the present invention, orotic acid is used in a higher yield in a culture liquid an inexpensive carbon source such as carbohydrates, organic acids and the like, and an inorganic one Formed nitrogen source as the starting material. By growing the mentioned microorganisms under suitable Conditions is almost only orotic acid in a considerable amount with the formation of only lower Amounts of by-products such as amino acids, organic acids and the like are accumulated. The educated The amount of orotic acid is so great that it far exceeds the solubility of the acid, its ammonium salt or other salts in the fermentation medium. This is how much of the orotic acid becomes or its salt is excreted in crystalline form in the culture medium during fermentation, and the

Process for biochemical production
of orotic acid

Applicant:

Kyowa Hakko Kogyo Co. Ltd., Tokyo

Representative:

Dr.-Ing. H. Ruschke, patent attorney,

Berlin 33, Auguste-Viktoria-Str. 65

Named as inventor:

Shukuo Kinoshita,

Katsunobu Tanaka, Tokyo;

Kazuo Kimura, Sagamihara-shi (Japan)

Claimed priority:

Japan June 15, 1960 (27 737)

Orotic acid formed can be separated and recovered in a simple manner after the cultivation is complete will.

The phenomenon that such a large amount of orotic acid in crystalline form in the culture medium has never been observed and is therefore new. So it became a beneficial novel Process for the industrial production of orotic acid found.

The micrococcus glutamicus strain according to the invention (534-615-305) ATCC 14275 needs uracil for growth and was obtained by artificial mutation of micrococcus glutamicus 534. the bacteriological properties of micrococcus glutamicus are in Japanese patent specification 243,382 and in Bulletin of the Agricultural Chemical Society of Japan, 22, No. 3, pp. 176-185. The mutant (534-615-305) ATCC 14275 was obtained by ultraviolet irradiation of the original strain and differs from this in that it needs uracil for growth.

All organic and synthetic culture media can be used in the fermentation process according to the invention

509 628/66

driving are used insofar as they are a carbon source, contain a nitrogen source, minerals, and other nutrients as shown in the following examples to be shown.

Various carbohydrates such as glucose, fructose, mannose, galactose, Sucrose, maltose, glycerin, mannitol, starch hydrolyzate, molasses and the like can be used. Further can organic acids, such as gluconic, vinegar, milk, pyrography, lemon, aconite, «ketoglutar, Fumaric, malic, oxaloacetic and other acids, alone or in combination with the carbohydrate be used.

As a nitrogen source, ammonia, various inorganic and organic ammonium salts, such as Ammonium sulfate, chloride, nitrate, phosphate, carbonate, acetate and the like, various nitrates, urea and other nitrogen-containing compounds such as peptone, NZ-amine, meat extract, yeast extract, Corn steep liquor, amino acids such as glutamic, aspartic and other acids, and hydrolysates of Proteins such as casein, fish meal, soybean grains, chrysalis, fermentation residues and others can be used. If a pure synthetic culture medium is used, an appropriate amount is added Biotin and uracil, which are necessary for the growth of bacteria, orotic acid in equal measure or in a much larger amount than when using the above-mentioned natural organic Nutrient.

As minerals dipotassium phosphate, monopotassium phosphate, magnesium sulfate, iron sulfate, manganese sulfate, Calcium carbonate and the like can be used.

Fermentation is preferably carried out under aerobic conditions, e.g. Example by cultivation with shaking culturing u with agitation and aeration. Like. The growth temperature may be 24 to 37 ⁰ C, preferably 28 to 33 ° C, respectively. The pH value tends to drop below 7.0 during growth, but it can preferably be adjusted to 5.5 to 8.5 using a suitable neutralizing agent at the beginning or during the cultivation if higher yields are to be achieved. As the neutralizing agent, aqueous ammonia, an alkali hydroxide such as sodium hydroxide, potassium hydroxide and the like, ammonium carbonate, calcium carbonate, calcium hydroxide and the like can be used. By adding urea, ammonia is supplied and a pH value is set that is favorable for the formation of orotic acid. A remarkably large amount of orotic acid accumulates in the culture medium within 2 to 3 days.

The present invention is illustrated in more detail by the following examples.

55 Example 1

In each of a number of 250 cc Erlenmeyer flasks were given 20 ecm of a fermentation medium and sterilized before use. The medium contains 5.0% glucose, 1.5% ammonium acetate, 0.05% monopotassium phosphate, 0.05% dipotassium phosphate, 0.5% urea, 0.5% yeast extract, 0.05% magnesium sulfate, 0.001% manganese sulfate, 0.5% ammonium sulfate and 10y / 1 biotin.

The bacterial mutants indicated below which require pyrimidines were each added to one of the media and ^{cultured at 30 °} C. with shaking.

After 72 hours, the amount of orotic acid formed was determined as follows:

Name of the bacterium Educated crowd
of orotic acid
(g / 100 ecm) Glutamic micrococcus
(534-615-305) ATCC 14275
Glutamic micrococcus
(588-208) ATCC 14276
Brevibacterium ammoniagenes
ATCC 6871 0.52
0.41
0.30

Example 2

In this example, the strain Micrococcus glutamicus (534-615-305) ATCC 14275 was used. 500 parts of a culture medium were placed in a 2000 ccm Erlenmeyer flask and before the Use sterilized.

The fermentation medium had the following composition: glucose 5.0%; KH ₂ PO ₄ 0.05%; K ₂ HPO ₄ 0.05%; MgSO ₄ · _{7H 2} O 0.05%; MnSO ₄ · 4 H ₂ O 0.001%; FeSO ₄ -7 H ₂ O 0.001%; (NH ^ ₂ SO ₄ 0.5%; urea 0.5%; uracil 100y / ccm; biotin 25y / l (pH 7.0).

After inoculating the strain, the medium was grown with shaking while keeping the temperature was kept at 30 ° C. During the cultivation, urea was added to the source of ammonia to supplement and to keep the pH of the fermentation liquid between 6.0 and 8.0. In the course of growth, crystals of ammonium orotate are separated from the medium. After 72 hours a liquid is obtained which contains 0.8% orotic acid.

Example 3

The procedure described in Example 2 was repeated using a culture medium containing 10.0% glucose, 0.05% KH ₂ PO ₄ , 0.05% K ₂ PO ₄ , 0.05% MgSO ₄ -7 H ₂ Contained 0.1001% MnSO ₄ -4H ₂ O, 0.001% FeSO _{4 -7H 2} O, 0.5% (NH 4 SO, 0.5% urea, 20 y / l biotin and 100 y / cc uracil. The cultivation lasted 72 hours. The amount of orotic acid formed was 10 mg / cc. The crystals precipitated during fermentation and were isolated, and the filtered fermentation liquor was concentrated. The crystals formed on cooling the concentrate were filtered off. From 11 fermentation liquor (which 10 g Orotic acid), ammonium orotate was isolated in an amount corresponding to 9.2 g of orotic acid The salt of orotic acid can easily be converted into the free acid by heating it with dilute hydrochloric acid.

Claims (1)

Claim: Process for the biochemical production of orotic acid by aerobic cultivation of a pyrimidine substance mutant microorganisms that require 5 6 ten in a nutrient medium customary for this purpose under glutamicus (588-208) ATCC 14276 or Brevi Maintaining a pH above 5.5, bacterium anunoniagenes ATCC 6871 used. characterized in that one the microorganism mutants Mikrokokkus glut- Considered publications: amicus (534-615-305) ATCC 14275, Micrococcus 5 USA. Patent No. 2,788,346. 509 628/66 7.65 © Bundesdruckerei Berlin